Polymyxin

Molecular formula: C 56 H 98 N 16 O 13 (B1)

Molecular weight: 1203.5(B 1 )

CAS Registry No.: 1406-11-7 (polymyxin), 1404-26-8 (polymyxin B), 1405-20-5 (polymyxin B sulfate)

SAMPLE
Matrix: formulations
Sample preparation: Sandwich cream or ointment between two layers of 200 mesh silica
gel, extract with carbon dioxide: MeOH 95:5 at 300 atmospheres at 55 at 2 mL/min for
75 min (restrictor 300), sonicate the SFE tube, frits, and silica gel with MeOH: 100 mM
HCl 25:75 containing 0.1% Tween 80 for 15 min, filter (0.2 jxm), inject an aliquot of the
filtrate. (SFE removes the hydrocarbon base of the cream or ointment leaving behind the
insoluble polymyxin.)
HPLCVARIABLES
Column: 250 X 4.6 5 |xm Synchropak SCD
Mobile phase: MeCN: buffer 21.5:78.5 (Buffer was 100 mM KH2PO4 containing 0.1% trifluoroacetic acid.)
Flow rate: 1.5
Injection volume: 50
Detector: UV 215
CHROMATOGRAM
Limit of quantitation: 0.016%
KEYWORDS
SFE; cream; ointment
REFERENCE
Moore, W.N.; Taylor, L.T. Analytical inverse supercritical fluid extraction of polar pharmaceutical compounds from cream and ointment matrices. J.Pharm.Biomed.AnaL, 1994, 12, 1227-1232

SAMPLE
Matrix: solutions
HPLCVARIABLES
Guard column: 45 X 4.7 Ultrasphere C18
Column: 250 X 4.7 Ultrasphere C18
Mobile phase: Gradient. A was 0.15% trifluoroacetic acid in water. B was 0.15% trifluoroacetic acid in MeCN. A:B from 100:0 to 50:50 over 25 min.
Flow rate: 2
Detector: UV 215
CHROMATOGRAM
Retention time: 18.5
OTHER SUBSTANCES
Simultaneous: degradation products
REFERENCE
Danner, R.L.; Joiner, K.A.; Rubin, M.; Patterson, W.H.; Johnson, N.; Ayers, K.M.; Parrillo, J.E. Purification, toxicity, and antiendotoxin activity of polymyxin B nonapeptide. Antimicrob.Agents Chemother., 1989, 33, 1428-1434

SAMPLE

Matrix: solutions
Sample preparation: Inject a 10 |xL aliquot of a 1 mg/mL solution.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Vydac TP C18

Mobile phase: Gradient. A was 0.1% trifluoroacetic acid in water. B was 0.075% trifluoroacetic acid in MeCN. A:B from 90:10 to 20:80 over 20 min.
Flow rate: 1.2
Injection volume: 10
Detector: UV 215
CHROMATOGRAM

Retention time: 12.0

OTHER SUBSTANCES

Simultaneous: degradation products.

REFERENCE
Vaara, M. Analytical and preparative high-performance liquid chromatography of the papain-cleaved
derivative of polymyxin B. J.Chromatogr., 1988, 441, 423-430

SAMPLE

Matrix: solutions
Sample preparation: Prepare a 400-500 |xg/mL solution in water, inject a 20 JULL aliquot.
HPLCVARIABLES

Column: 150 X 4.6 5 |xm Nucleosil 5 C18

Mobile phase: MeCN: buffer 23:77 (Buffer was 23 mM phosphoric acid containing 10 mM
acetic acid and 50 mM sodium sulfate, adjust pH to 2.5 with triethylamine.)
Flow rate: 0.9
Injection volume: 20
Detector: UV 220
CHROMATOGRAM

Retention time: 8 (polymyxin B2), 15 (polymyxin Bl)

OTHER SUBSTANCES

Also analyzed: colistin (polymyxin E)

REFERENCE
Elverdam, L; Larsen, P.; Lund, E. Isolation and characterization of three new polymyxins in polymyxins
B and E by high-performance liquid chromatography. J.Chromatogr., 1981, 218, 653-661

SAMPLE

Matrix: solutions
Sample preparation: Prepare a 10-100 [xg/wiL solution in mobile phase, inject an aliquot.
HPLCVARIABLES

Column: 250 X 4.6 5 |xm Ultrasphere ion-pair

Mobile phase: MeCN: buffer 23:77 (Prepare mobile phase by mixing 230 mL MeCN, 700
mL water, and 38 g Na3PO4, adjust pH to 3.0 with phosphoric acid, make up to 1 L with
water.)
Column temperature: 27
Flow rate: 1

Injection volume: 10
Detector: UV 185; UV 200
CHROMATOGRAM
Retention time: 7 (B2), 8 (B3), 13 (Bl)
Limit of detection: 30 ng (UV 185)
OTHER SUBSTANCES
Simultaneous: colistin
REFERENCE
Whall, T.J. High-performance liquid chromatography of polymyxin B sulfate and colistin sulfate.
J.Chromatogr., 1981,208, 118-123

SAMPLE
Matrix: solutions
Sample preparation: Inject a 5-50 \xL aliquot of a 1 mg/mL solution in water.
HPLCVARIABLES
Column: 200 X 4 Nucleosil 5C18
Mobile phase: MeCN: buffer 22.5:77.5 (Buffer was 5 mM pH 3.0 tartrate buffer containing
5 mM sodium 1-butanesulfonate and 50 mM sodium sulfate.)
Flow rate: 1
Injection volume: 5-50
Detector: UV 220
CHROMATOGRAM
Retention time: 11 (polymyxin B2), 13 (polymyxin B3), 23.5 (polymyxin B1)
OTHER SUBSTANCES
Simultaneous: colistin, polymyxin C, polymyxin D, polymyxin E, polymyxin M, polymyxin
S, polymyxin T
REFERENCE
Terabe, S.; Konaka, R.; Shoji, J. Separation of polymyxins and octapeptins by high-performance liquid
chromatography. J.Chromatogr., 1979, 173, 313-320

ANNOTATED BIBLIOGRAPHY
Kitamura-Matsunaga, H.; Kimura, Y.; Araki, T.; Izumiya, N. A sodium-containing polymyxin derived
from polymyxin-complex during chromatography. J.Antibiot.(Tokyo), 1984, 37, 1605-1610 [for colistin, polymyxin E; analytical; preparative]